Effect of nitrogen forms and spikelet positions on endophyte

A range of histochemical, immunological and molecular methods have been used to detect endophyte in vegetative plant tissues and seed tissues. In this project, the immunoblot test was applied to determine the viability of endophyte. The method is a common test for endophyte present and fit for the purpose for this study.

3.5.1. Immunoblot detection of endophyte 3.5.1.1. Growing the seedlings for the assay

Cultivar ‘Halo’ AR37 seeds or transplanted seedlings from the germination test (if insufficient seed was available, i.e. replicate 2 of treatment 2, replicate 2 of treatment 4, and the top spikelet position of replicate 3 of treatment 5) were grown on in 54 hygiene trays with 96 seedlings in each tray (dimensions: 30 cm wide, 42 cm long and 6 cm high). Seedlings were grown in a glasshouse with a diurnal temperature range of 16 °C to 26 °C located at the Massey University Plant Growth

Unit (40o22’S, 175o36’E). Seedlings were grown in a potting medium consisting of

peat (333 litres), bark (867 litres), coir fibre (200 litres), pumice (400 litres) and sterilised river sand (200 litres), plus 4 kg 3-4 month of Osmocote (N-P-K: 16-9-12 plus 2% MgO plus trace elements) (Figure 13). Each tray was filled with potting medium and a 96 station (position) stamp that fitted neatly inside the hygiene tray was used to imprint 96 holes in the medium in each tray. A seed or seedling was sown by hand into each station hole to allow up to 96 plants per tray. Additional potting medium was applied to cover the seeds in the holes and the trays were watered and placed in the glasshouse. Each tray was labelled and daily overhead watering was applied until the plant had grown three to four tillers (from 26 August to 10 October 2013) before the immunoblot test was conducted.

Figure 13. Proportional components of potting medium. Note: 4 kg/2000 litres 3-4 month Osmocote fertiliser was also included in the mix.

Figure 14. Top and bottom views of a stamp with 96 stations, used to sow seeds or seedlings into seedling trays for later immunoblot analysis.

3.5.1.2. Blotting the endophyte

Two tillers per plant were carefully cut at the tiller base where the sheath tissue is located. The fresh cut end of each tiller was blotted onto nitrocellulose membrane

333 ltrs 867 ltrs 200 ltrs 400 ltrs 200 ltrs

Potting medium

sheets leaving circular outlines (from the compressed pseudostem leaves) of the cut ends. The blot was assessed as positively or negatively stained for endophyte.

3.5.1.3. Staining the endophyte

A blocking solution was used to reduce the background staining by blocking reactive sites where the primary or secondary antibody may bind. The amount of blocking solution required per sheet was calculated to be about 25 ml. The blocking solution was prepared by dissolving 2.42 g Tris (hydroxymethyl) methylamine, 2.92 g NaCl, 5 g non-fat milk powder, and 10 ml of 1 mol HCl before being made up to one litre using reverse osmosis (RO) water. The RO water was measured and poured into a container, then the dry ingredients were added first and then the measured volume of HCl was added to make a final volume of one litre. Finally, the pH of the blocking solution was adjusted to pH 7.5 by adding concentrated HCl. The blotted sheets with no bound protein were immersed in the blocking solution and membrane sheet, then, shaken

on an orbital shaker at room temperature (approximately 20°C) for at least 2 hours.

The old dark solution was poured out and fresh blocking solution added into the

container using 25 ml of blocking solution per sheet. Primary antibody (25 μl per

sheet) was then added to the blocking solution and the sheets were left on the shaker overnight at 4 °C. The following day membrane sheets were rinsed three times with the fresh blocking solution and then 25 ml per sheet of fresh blocking

solution was added with 6.25 μl per sheet of secondary antibody. The membrane

was shaken at room temperature for at least 2 hours. Tris buffer was prepared by dissolving 24.2 g of Tris (hydroxymethyl) methylamine per litre RO water and the pH value adjusted to 8.2. The Tris buffer was then put on a shaker for a couple of minutes. Fast Red (20 mg per sheet) and Napthol (12.5 mg per sheet) were dissolved in 12.5 ml Tris buffer separately and then the two solutions were combined to produce a chromogen solution. The blocking solution was decanted off; the sheets were rinsed twice with fresh blocking solution before the chromogen solution was

poured over the membrane sheets. The membrane sheets with chromogen solution were shaken at room temperature for 15 minutes until blots developed. Finally, the sheets were rinsed three times with RO water and then rinsed with tap water. The developed sheets were placed on a paper towel and then assessed where a bright red colour indicated a positive blot and a light red colour indicated a negative blot

(Simpson et al., 2012). After tillers were taken for the assay the remaining plant was

placed outside the glasshouse at AgResearch, Palmerston North (Figure 15) to provide further tillers for re-blotting when it was not clear from the immunoblot if endophyte was present or not.

Figure 15. Lolium plants kept outside the glasshouse at AgResearch Grasslands, Palmerston North in case needed for endophyte reassessment.